EP0090735A2
Expired / Lapsed
EP
⏱️ 0.0 years remaining
Lupus assay method and compositions
Assignee: Elan Pharmaceuticals LLC
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Filing: 1983-03-25
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Granted: —
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Expires: 2003-03-25
Abstract
Abstract An assay for detecting SLE antibodies utilizing a novel liposome composition in which there is entrapped a divalent cation responsive indicator. The presence of such antibodies is detected by their stabilization of the supermolecular structure of the liposomes. Such stabilization can be detected, and in certain cases, caused by, the addition of magnesium or calcium ions.
Inventors
- 👤 Andrew Stuart Janoff
- 👤 Marc J. Ostro
- 👤 Allan L. Weiner
- 👤 Gerald Weissmann
- 👤 James R. Seibold
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-08 23:36
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/564
Claims Analysis
Total Claims: 53
Claim 1
Independent Claim
113 words
A method for detecting SLE antibodies in serum, comprising: (a) contacting serum, for a period of time at least sufficient to permit equilibration of binding between SLE antibodies and cardiolipin, with liposomes having a multicomponent liposome bilayer comprising a flexible double tailed phosphatidylcholine and cardiolipin in a molar ratio of from about 1:7 to about 7:1, respectively; (b) exposing said liposomes to conditions which cause destablization of the liposomes in the absence of SLE antibodies and which do not cause destablization of the liposomes in the presence SLE antibodies; and (c) detecting any destablization of the liposomes, said method being performed under conditions non-inhibiting to the binding between SLE antibodies and said cardiolipin.
Claim 2
Independent Claim
113 words
A method for detecting SLE antibodies in serum, comprising: (a) contacting serum, for a period of time at least sufficient to permit equilibration of binding between SLE antibodies and cardiolipin, with liposomes having a multicomponent liposome bilayer comprising a flexible double tailed phosphatidylcholine and cardiolipin in a molar ratio of from about 1:7 to about 7:1, respectively; (b) exposing said liposomes to conditions which cause destablization of the liposomes in the absence of SLE antibodies and which do not cause destablization of the liposomes in the presence SLE antibodies; and (c) detecting any destablization of the liposomes, said method being performed under conditions non-inhibiting to the binding between SLE antibodies and said cardiolipin.
Claim 3
Dependent Claim
16 words
The method according to claim 1, wherein said ratio is from about 2:5 to about 4:3.
Claim 4
Dependent Claim
42 words
The method according to claim 1, wherein said liposomes contain entrapped therein a divalent cation responsive colorimetric indicator, and said conditions causing destablization comprise contacting said liposomes with divalent cations which results in leakage and a change in color of the indicator.
Claim 5
Dependent Claim
15 words
The method according to claim 3, wherein the divalent cations are magnesium or calcium cations.
Claim 6
Independent Claim
112 words
A method for detecting SLE antibodies in serum, comprising: (a) contacting serum, for a period time sufficient to permit equilibration of binding between SLE antibodies and a phosphatidic acid antigen which binds to SLE antibodies, with liposomes having a multicomponent liposome bilayer comprising a flexible double tailed phosphatidylcholine and said phosphatidic acid antigen in molar ratios of from about 9:1 to about 3:7, respectively, said liposomes being unstable in the presence of calcium ions unless SLE antibodies are present; (b) exposing said liposomes to calcium ions; and (c) detecting any destabilization of the liposomes, said method being performed under conditions non-inhibiting to the binding between SLE antibodies and said phosphatidic acid antigen.
Claim 7
Independent Claim
112 words
A method for detecting SLE antibodies in serum, comprising: (a) contacting serum, for a period time sufficient to permit equilibration of binding between SLE antibodies and a phosphatidic acid antigen which binds to SLE antibodies, with liposomes having a multicomponent liposome bilayer comprising a flexible double tailed phosphatidylcholine and said phosphatidic acid antigen in molar ratios of from about 9:1 to about 3:7, respectively, said liposomes being unstable in the presence of calcium ions unless SLE antibodies are present; (b) exposing said liposomes to calcium ions; and (c) detecting any destabilization of the liposomes, said method being performed under conditions non-inhibiting to the binding between SLE antibodies and said phosphatidic acid antigen.
Claim 8
Dependent Claim
36 words
The method according to claim 5, wherein said phosphatidic acid is dipalmitoylphosphatidic acid, and said liposomes have a multicomponent liposome bilayer comprising egg phosphatidylcholine, dipalmitoylphosphatidic acid, cholesterol and dicetylphosphate in molar ratios of about 7:1:1:1, respectively.
Claim 9
Dependent Claim
41 words
The method according to claim 5, wherein said liposomes contain entrapped therein a divalent cation responsive colorimetric indicator, and said destablization results in an influx of divalent cations into the liposomes which results in a change in color of the indicator.
Claim 10
Independent Claim
115 words
A method for detecting SLE antibodies in serum, comprising: (a) contacting serum, for a period of time at least sufficient to permit equilibration of binding between SLE antibodies and a phosphatidic acid antigen which binds to SLE antibodies, with liposomes having a multicomponent liposome bilayer comprising a flexible double tailed phosphatidylcholine and said phosphatidic acid antigen in a molar ratio of from about 1:7 to about 7:1, respectively, said liposomes being stable in the presence of serum containing SLE antibodies and unstable in the presence of serum not containing SLE antibodies; and (b) detecting any liposome instability, said method being performed under conditions non-inhibiting to the binding between SLE antibodies and said phosphatidic acid antigen.
Claim 11
Independent Claim
115 words
A method for detecting SLE antibodies in serum, comprising: (a) contacting serum, for a period of time at least sufficient to permit equilibration of binding between SLE antibodies and a phosphatidic acid antigen which binds to SLE antibodies, with liposomes having a multicomponent liposome bilayer comprising a flexible double tailed phosphatidylcholine and said phosphatidic acid antigen in a molar ratio of from about 1:7 to about 7:1, respectively, said liposomes being stable in the presence of serum containing SLE antibodies and unstable in the presence of serum not containing SLE antibodies; and (b) detecting any liposome instability, said method being performed under conditions non-inhibiting to the binding between SLE antibodies and said phosphatidic acid antigen.
Claim 12
Dependent Claim
16 words
The method according to claim 8, wherein said ratio is from about 2:5 to about 4:3.
Claim 13
Dependent Claim
14 words
The method according to claim 8, wherein said phosphatidic acid is l-palmitoyl-2-oleoyl- phosphatidic acid.
Claim 14
Dependent Claim
40 words
The method according to claim 8, wherein said liposomes contain entrapped therein a colorimetric indicator, and said instability is detected by a color change in said indicator which is released from the liposomes in the absence of SL E antibodies.
Claim 15
Dependent Claim
26 words
The method according to claim 11, wherein said indicator is responsive to divalent metal ions and is exposed to divalent cations upon release from the liposomes.
Claim 16
Dependent Claim
14 words
The method according to claim 12, wherein the divalent metal ions are magnesium ions.
Claim 17
Dependent Claim
22 words
The method according to claim 1, 5 or 8, wherein the anionic lipid components of the bilayer are cesium or potassium salts.
Claim 18
Dependent Claim
15 words
The method according to claim 1, 5 or 8, wherein said phosphatidylcholine is l-palmitoyl-2-oleoyl- phosphatidylcholine.
Claim 19
Dependent Claim
22 words
The method according to claim 1, 5 or 8, wherein said liposome bilayer contains a stabilizing amount of a rigid steriod stabilizer.
Claim 20
Dependent Claim
11 words
The method according to claim 16, wherein said steroid is cholesterol.
Claim 21
Dependent Claim
32 words
The method according to claim 17, wherein the stabilizing amount is from about 5 to about 30 molar percent, relative to the total amount of said phosphatidylcholine and phosphatidic acid or cardiolipin.
Claim 22
Dependent Claim
41 words
The method according to claim 1 or 8, wherein said liposome bilayer contains from about 10 to about 20 molar percent, relative to the total amount of said phosphatidylcholine and phosphatidic acid or cardiolipin, of a liposome compatible negatively charged compound.
Claim 23
Dependent Claim
15 words
The method according to claim 19, wherein said liposome compatible negatively charged compound is phosphatidylglycerol.
Claim 24
Dependent Claim
11 words
The method according to claim 20, wherein said phosphatidylglycerol is dipalmitoyl-phosphatidylglycerol.
Claim 25
Dependent Claim
31 words
The method according to claim 5, wherein said liposome bilayer contains from about 10 to about 20 molar percent, relative to the total amount of phosphatidylcholine and phosphatidic acid, of dicetylphosphate.
Claim 26
Dependent Claim
20 words
The method according to claim 1, 5 or 8, wherein said liposome bilayer contains a stabilizing amount of an antioxidant.
Claim 27
Dependent Claim
36 words
The method according to claim 23, wherein said antioxidant is alpha-tocopherol in an amount corresponding to from about 1 to about 5 molar percent of the total amount of said phosphatidylcholine and phosphatidic acid or cardiolipin.
Claim 28
Dependent Claim
26 words
The method according to claim 1, wherein said bilayer comprises l-palmitoyl-2-oleoyl- phosphatidylcholine, cardiolipin, cholesterol, alpha-tocopherol and dipalmitoyl phosphatidylglycerol in a molar ratio of about 3:4:1.9:0.1:1, respectively.
Claim 29
Dependent Claim
27 words
The method according to claim 8, wherein said bilayer comprises 1-palmitoyl-2-oleoyl- phosphatidylcholine, l-palmitoyl-2-oleoyl-phosphatidic acid, cholesterol, alpha-tocopherol and dipalmitoyl phosphatidylglycerol in a molar ratio of about 3:4:1.9:0.1:1, respectively.
Claim 30
Dependent Claim
15 words
The method according to claim 1, 5 or 8, wherein said indicator is arsenazo III.
Claim 31
Dependent Claim
48 words
A liposome composition comprising liposome vesicles, said vesicles having (i) a multicomponent liposome bilayer comprising a flexible double tailed phosphatidylcholine and a lipid antigen for SLE antibodies in a molar ratio of from about 1:7 to about 9:1, respectively, and (ii) a colorimetric indicator entrapped within said liposome.
Claim 32
Dependent Claim
48 words
A liposome composition comprising liposome vesicles, said vesicles having (i) a multicomponent liposome bilayer comprising a flexible double tailed phosphatidylcholine and a lipid antigen for SLE antibodies in a molar ratio of from about 1:7 to about 9:1, respectively, and (ii) a colorimetric indicator entrapped within said liposome.
Claim 33
Dependent Claim
16 words
A composition according to claim 28, wherein said indicator is a divalent cation responsive colorimetric indicator.
Claim 34
Dependent Claim
16 words
A composition according to claim 28, wherein said ratio is from about 2:5 to about 4:3.
Claim 35
Dependent Claim
16 words
A composition according to claim 28, wherein said lipid antigen is a phosphatidic acid or cardiolipin.
Claim 36
Dependent Claim
12 words
A composition according to claim 28, wherein said phosphatidylcholine is l-palimitoyl-2-oleoyl- phosphatidylcholine.
Claim 37
Dependent Claim
28 words
A composition of claim 28, wherein said vesicles have a multicomponent liposome bilayer comprising egg phosphatidylcholine, dipalmitoylphosphatidic acid, cholesterol and dicetylphosphate in molar ratios of about 7:1:1:1, respectively.
Claim 38
Dependent Claim
19 words
A composition according to claim 28, wherein said liposome bilayer contains a stabilizing amount of a rigid steroid stabilizer.
Claim 39
Dependent Claim
11 words
A composition according to claim 34, wherein said steriod is cholesterol.
Claim 40
Dependent Claim
32 words
A composition accoraing to claim 34, wherein the stabilizing amount is from about 5 to about 30 molar percent, relative to the total of amount of said phosphatidylcholine and said lipid antigen.
Claim 41
Dependent Claim
37 words
A composition according to claim 28, wherein said liposome bilayer contains from about 10 to about 20 molar percent, relative to the total of said phosphatidylcholine and said lipid antigen, of a liposome compatible negatively charged compound.
Claim 42
Dependent Claim
16 words
A composition according to claim 37, wherein said liposome compatible negatively charged compound is a phosphatidylglycerol.
Claim 43
Dependent Claim
12 words
A composition according to claim 38, wherein said phosphatidylglycerol is dipalmitoyl- phosphatidylgylycerol.
Claim 44
Dependent Claim
17 words
A composition according to claim 28, wherein said liposome bilayer contains a stabilizing amount of an antioxidant.
Claim 45
Dependent Claim
35 words
A composition according to claim 40, wherein said antioxidant is alpha-tocopherol in an amount corresponding to from about 1 to about 5 molar percent of the total amount of said phosphatidylcholine and said lipid antigen.
Claim 46
Dependent Claim
24 words
A composition according to claim 28, wherein said liposome bilayer comprises egg phosphatidylcholine, dipalmitoylphosphatidic acid, cholesterol and dicetylphosphate in molar ratios of 7:1:1:1, respectively.
Claim 47
Dependent Claim
27 words
A composition according to claim 28, wherein said liposome bilayer comprises l-palmitoyl-2-oleoyl- phosphatidylcholine, cardiolipin, cholesterol, alpha-tocopherol and dipalmitoyl phosphatidylglycerol in a molar ratio of about 3:4:1.9:0.1:1, respectively.
Claim 48
Dependent Claim
27 words
A composition according to claim 28, wherein said liposome bilayer comprises 1-palmitoyl-2-oleoyl- phosphatidylcholine, l-palmitoyl-2-oleoyl-phosphatidic acid, cholesterol, alpha-tocopherol and dipalmitoylphosphatidylglycerol in a molar ratio of about 3:4:1.9:0.1:1, respectively.
Claim 49
Dependent Claim
14 words
A composition according to claim 28 or 43, wherein said indicator is arsenazo III.
Claim 50
Dependent Claim
16 words
A composition according to claim 28, wherein said lipid antigen is a cesium or potassium salt.
Claim 51
Dependent Claim
28 words
A composition according to claim 28, wherein said lipid antigen is the cesium salt of cardiolipin and wherein said liposome bilayer further comprises the potassium salt of dipalmitoylphosphatidylglycerol.
Claim 52
Independent Claim
102 words
A method for preparing liposomes having a compound entrapped therein, comprising: (a) forming a dispersion of at least one amphipathic lipid in an organic solvent; (b) forming an aqueous mixture containing a compound to be entrapped in the liposomes; - (c) injecting the dispersion into the aqueous mixture heated to a temperature above the boiling point of the organic solvent to form, as the organic solvent escapes, a suspension of liposomes having said compounds entrapped therein; and (d) exposing the suspension to a vacuum produced by a water aspirator for from about one to five hours to remove excess remaining organic solvent.
Claim 53
Independent Claim
102 words
A method for preparing liposomes having a compound entrapped therein, comprising: (a) forming a dispersion of at least one amphipathic lipid in an organic solvent; (b) forming an aqueous mixture containing a compound to be entrapped in the liposomes; - (c) injecting the dispersion into the aqueous mixture heated to a temperature above the boiling point of the organic solvent to form, as the organic solvent escapes, a suspension of liposomes having said compounds entrapped therein; and (d) exposing the suspension to a vacuum produced by a water aspirator for from about one to five hours to remove excess remaining organic solvent.
References Cited (Backward Citations)
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